Chemically cleavable group

Abstract

Disclosed is the use of the reactive components of the inverse electron-demand Diels Alder reaction for chemical masking and unmasking in vitro. This can be applied in complex chemical reactions and, particularly in the synthesis of biomolecules, e.g. on solid supports. The reactice components are a dienophile, particularly a trans-cyclooctene, and a diene, particularly a tetrazine.

1 . A dienophile as a chemically cleavable group attached to a Construct, thereby provoking the release, in vitro, of the Construct by allowing the dienophile to react with a diene, wherein the dienophile is an eight-membered non-aromatic cyclic alkenylene group, preferably a cyclooctene group, and more preferably a trans-cyclooctene group. 
     
     
         2 . The dienophile according to  claim 1 , wherein the dienophile satisfies Formula (1a): 
       
         
           
           
               
               
           
         
         wherein A and P each independently are CR a   2  or CR a X D , provided that at least one is CR a X D . X D  is (O—C(O)) p -(L D ) n -(C A ), S—C(O)-(L D ) n -(C A ), O—C(S)-(L D ) n -(C A ), S—C(S)-(L D ) n -(C A ), O—S(O)-(L D ) n -(C A ), wherein p=0 or 1. Preferably, X D  is (O—C(O)) p -(L D ) n -(C A ), where p=0 or 1, preferably 1, and n=0 or 1; Y,Z,X,Q each independently are selected from the group consisting of CR a   2 , C═CR a   2 , C═O, C═S, C═NR b , S, SO, SO 2 , O, NR b , and SiR c   2 , with at most three of Y, Z, X, and Q being selected from the group consisting of C═CR a   2 , C═O, C═S, and C═NR b , wherein two R moieties together may form a ring, and with the proviso that no adjacent pairs of atoms are present selected from the group consisting of O—O, O—NR b , S—NR b , O—S, O—S(O), O—S(O) 2 , and S—S, and such that Si is only adjacent to CR a   2  or O. 
       
     
     
         3 . The dienophile according to  claim 1 , wherein the dienophile satisfies Formula (1a): 
       
         
           
           
               
               
           
         
         wherein A and P each independently are CR a   2  or CR a X D , provided that at least one is CR a X D . X D  is (O—C(O)) p -(L D ) n -(C A ), S—C(O)-(L D ) n -(C A ), O—C(S)-(L D ) n -(C A ), S—C(S)-(L D ) n -(C A ), O—S(O)-(L D ) n -(C A ), wherein p=0 or 1. Preferably, X D  is (O—C(O)) p -(L D ) n -(C A ), where p=0 or 1, preferably 1, and n=0 or 1; wherein one of the bonds PQ, QX, XZ, ZY, YA is part of a fused ring or consists of CR a ═CR a , such that two exocyclic bonds are fixed in the same plane, and provided that PQ and YA are not part of an aromatic 5- or 6-membered ring, of a conjugated 7-membered ring, or of CR a ═CR a ; when not part of a fused ring P and A are independently CR a   2  or CR a X D , provided that at least one is CR a X D ; when part of a fused ring P and A are independently CR a  or CX D , provided that at least one is CX D ; the remaining groups (Y,Z,X,Q) being independently from each other CR a   2 , C═CR a   2 , C═O, C═S, C═NR b , S, SO, SO 2 , O, NR b , SiR c   2 , such that at most 1 group is C═CR a   2 , C═O, C═S, C═NR b , and no adjacent pairs of atoms are present selected from the group consisting of O—O, O—NR b , S—NR b , O—S, O—S(O), O—S(O) 2 , and S—S, and such that Si, if present, is adjacent to CR a   2  or O, and the CR a   2 ═CR a   2  bond, if present, is adjacent to CR a   2  or C═CR a   2  groups. 
       
     
     
         4 . The dienophile according to  claim 1 , wherein the dienophile comprises a treats cyclooctene moiety that satisfies formula (1b): 
       
         
           
           
               
               
           
         
         wherein, in addition to the optional presence of at most two exocyclic bonds fixed in the same plane, each R a  independently denotes H, or, in at most four instances, a substituent selected from the group consisting of alkyl, aryl, OR′, SR′, S(═O)R′″, S(═O) 2 R′″, S(═O) 2 NR′R″, Si—R′″, Si—O—R′″, OC(═O)R′″, SC(═O)R′″, OC(═S)R′″, SC(═S)R′″, F, Cl, Br, I, N 3 , SO 2 H, SO 3 H, SO 4 H, PO 3 H, PO 4 H, NO, NO 2 , CN, OCN, SCN, NCO, NCS, CF 3 , CF 2 —R′, NR′R″, C(═O)R′, C(═S)R′, C(═O)O—R′, C(═S)O—R′, C(═O)S—R′, C(═S)S—R′, C(═O)NR′R″, C(═S)NR′R″, NR′C(═O)—R′″, NR′C(═S)—R′″, NR′C(═O)O—R′″, NR′C(═S)O—R′″, NR′C(═O)S—R′″, NR′C(═S)S—R′″, OC(═O)NR′—R′″, SC(═O)NR′—R′″, OC(═S)NR′—R′″, SC(═S)NR′—R′″, NR′C(═O)NR″—R″, NR′C(═S)NR″—R″, CR′NR″, with each R′ and each R″ independently being H, aryl or alkyl and R′″ independently being aryl or alkyl; 
         wherein each R e  as above indicated is independently selected from the group consisting of a alkyl, aryl, OR′, SR′, S(═O)R″ S(═O) 2 R″ Si—R′″, Si—O—R′″, OC(═O)R″ SC(═O)R′″, OC(═S)R′″, SC(═S)R′″, F, Cl, Br, I, N 3 , SO 2 H, SO 3 H, PO 3 H, NO, NO 2 , CN, CF 3 , CF 2 —R′, C(═O)R′, C(═S)R′, C(═O)O—R′, C(═S)O—R′, C(═O)S—R′, C(═S)S—R′, C(═O)NR′R″, C(═S)NR′R″, NR′C(═O)—R′″, NR′C(═S)—R″ NR′C(═O)O—R″ NR′C(═S)O—R′″, NR′C(═O)S—R′″, NR′C(═S)S—R′″, NR′C(═O)NR″—R″, NR′C(═S)NR″—R″, CR′NR″, with each R′ and each R″ independently being a aryl or alkyl and R′″ independently being aryl or alkyl; 
         wherein two R a,e  moieties together may form a ring; 
         wherein optionally one R a,e  or the self-immolative linker L D , is bound, optionally via a spacer or spacers S P , to C B , and wherein T and F each independently denote a or a substituent selected from the group consisting of alkyl, F, Cl, Br, and I, and X D  is (O—C(O)) p -(L D ) n -(C A ), S—C(O)-(L D ) n -(C A ), O—C(S)-(L D ) n -(C A ), S—C(S)-(L D ) n -(C a ), O—S(O)-(L D ) n -(C A ), wherein p=0 or 1. 
       
     
     
         5 . The dienophile according to  claim 1 , wherein the trans-cyclooctene moiety satisfies any one of the following formulae: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         6 . The dienophile according to  claim 1 , wherein the trans-cyclooctene moiety satisfies any one of the following formulae: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         7 . The dienophile according to  claim 1 , wherein the trans-cyclooctene moiety satisfies any one of the following formulae 
       
         
           
           
               
               
           
         
       
     
     
         8 . The dienophile according to  claim 1 , wherein the trans-cyclooctene moiety satisfies any one of the following formulae 
       
         
           
           
               
               
           
         
       
     
     
         9 . The dienophile according to  claim 1 , wherein the trans-cyclooctene moiety satisfies any one of the following formulae 
       
         
           
           
               
               
           
         
       
     
     
         10 . The dienophile according to  claim 1 , wherein the diene satisfies any one of the following formulae (2) to (4): 
       
         
           
           
               
               
           
         
         wherein R 1  is selected from the group consisting of H, alkyl, aryl, CF 3 , CF 2 —R′, OR′, SR′, C(═O)R′, C(═S)R′, C(═O)O—R′, C(═O)S—R′, C(═S)O—R′, C(═S)S—R″, C(═O)NR′R″, C(═S)NR′R″, NR′R″, NR′C(═O)R″, NR′C(═S)R″, NR′C(═O)OR″, NR′C(═S)OR″, NR′C(═O)SR″, NR′C(═S)SR″, NR′C(═O)NR″R″, and NR′C(═S)NR″R″ with each R′ and each R″ independently being H, aryl or alkyl; A and B each independently are selected from the group consisting of alkyl-substituted carbon, aryl substituted carbon, nitrogen, N + O − , N + R with R being alkyl, with the proviso that A and B are not both carbon; X is selected from the group consisting of O, N-alkyl, and C═O, and Y is CR with R being selected from the group consisting of a alkyl, aryl, C(═O)OR′, C(═O)SR′, C(═S)OR′, C(═S)SR′, C(═O)NR′R″ with R′ and R″ each independently being H, aryl or alkyl; 
       
       
         
           
           
               
               
           
         
         wherein R 1  and R 2  each independently are selected from the group consisting of H, alkyl, aryl, CF 3 , CF 2 —R′, NO 2 , OR′, SR′, C(═O)R′, C(═S)R′, OC(═O)R″ SC(═O)R′″, OC(═S)R′″, SC(═S)R′″, S(═O)R′, S(═O) 2 R′″, S(═O) 2 NR′R″, C(═O)O—R′, C(═O)S—R′, C(═S)O—R′, C(═S)S—R′, C(═O)NR′R″, C(═S)NR′R″, NR′R″, NR′C(═O)R″, NR′C(═S)R″, NR′C(═O)OR″, NR′C(═S)OR″, NR′C(═O)SR″, NR′C(═S)SR″, OC(═O)NR′R″, SC(═O)NR′R″, OC(═S)NR′R″, SC(═S)NR′R″, NR′C(═O)NR″R″, and NR′C(═S)NR″R″ with each R′ and each R″ independently being H, aryl or alkyl, and R′″ independently being aryl or alkyl; A is selected from the group consisting of N-alkyl, N-aryl, C═O, and CN-alkyl; B is O or S; X is selected from the group consisting of N, CH, C-alkyl, C-aryl, CC(═O)R′, CC(═S)R′, CS(═O)R′, CS(═O) 2 R′″, CC(═O)O—R′, CC(═O)S—R′, CC(═S)O—R′, CC(═S)S—R′, CC(═O)NR′R″, and CC(═S)NR′R″, R′ and R″ each independently being H, aryl or alkyl and R′″ independently being aryl or alkyl; Y is selected from the group consisting of CH, C-alkyl, C-aryl, N, and N + O − . 
       
       
         
           
           
               
               
           
         
         wherein R 1  and R 2  each independently are selected from the group consisting of H, alkyl, aryl, CF 3 , CF 2 —R′, NO, NO 2 , OR′, SR′, CN, C(═O)R′, C(═S)R′, OC(═O)R′″, SC(═O)R′″, OC(═S)R′″, SC(═S)R′″, S(═O)R′, S(═O) 2 R′″, S(═O) 2 OR′, PO 3 R′R″, S(═O) 2 NR′R″, C(═O)O—R′, C(═O)S—R′, C(═S)O—R′, C(═S)S—R′, C(═O)NR′R″, C(═S)NR′R″, NR′R″, NR′C(═O)R″, NR′C(═S)R″, NR′C(═O)OR″, NR′C(═S)OR″, NR′C(═O)SR″, NR′C(═S)SR″, OC(═O)NR′R″, SC(═O)NR′R″, OC(═S)NR′R″, SC(═S)NR′R″, NR′C(═O)NR″R″, and NR′C(═S)NR″R″ with each R′ and each R″ independently being H, aryl or alkyl, and R′″ independently being aryl or alkyl; A is selected from the group consisting of N, C-alkyl, C-aryl, and N + O − ; B is N; X is selected from the group consisting of N, CH, C-alkyl, C-aryl, CC(═O)R′, CC(═S)R′, CS(═O)R′, CS(═O) 2 R′″, CC(═O)O—R′, CC(═O)S—R′, CC(═S)O—R′, CC(═S)S—R′, CC(═O)NR′R″, CC(═S)NR′R″, R′ and R″ each independently being H, aryl or alkyl and R′″ independently being aryl or alkyl; Y is selected from the group consisting of CH, C-alkyl, C-aryl, N, and N + O − . 
       
     
     
         11 . The dienophile according to  claim 10 , wherein the diene satisfies formula (7) as defined in the description. 
     
     
         12 . The dienophile according to  claim 10  wherein the diene satisfies formula (8a) or (8b): 
       
         
           
           
               
               
           
         
         wherein each R 1  and each R 2  independently are selected from the group consisting of H, alkyl, aryl, CF 3 , CF 2 —R′, NO 2 , OR′, SR′, C(═O)R′, C(═S)R′, OC(═O)R′″, SC(═O)R′″, OC(═S)R′″, SC(═S)R′″, S(═O)R′, S(═O) 2 R′″, S(═O) 2 NR′R″, C(═O)O—R′, C(═O)S—R′, C(═S)O—R′, C(═S)S—R′, C(═O)NR′R″, C(═S)NR′R″, NR′R″, NR′C(═O)R″, NR′C(═S)R″, NR′C(═O)OR″, NR′C(═S)OR″, NR′C(═O)SR″, NR′C(═S)SR″, OC(═O)NR′R″, SC(═O)NR′R″, OC(═S)NR′R″, SC(═S)NR′R″, NR′C(═O)NR″R″, and NR′C(═S)NR″R″ with each R′ and each R″ independently being H, aryl or alkyl, and R′″ independently being aryl or alkyl. 
       
     
     
         13 . The dienophile according to  claim 10 , wherein the diene is satisfies a formula selected from the group consisting of (8c), (8d), (8e), (8f), and (8g): 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         wherein each R 1  and each R 2  independently are selected from the group consisting of H, alkyl, aryl, CF 3 , CF 2 —R′, NO 2 , OR′, SR′, C(═O)R′, C(═S)R′, OC(═O)R′″, SC(═O)R′″, OC(═S)R′″, SC(═S)R′″, S(═O)R′, S(═O) 2 R′″, S(═O) 2 NR′R″, C(═O)O—R′, C(═O)S—R′, C(═S)O—R′, C(═S)S—R′, C(═O)NR′R″, C(═S)NR′R″, NR′R″, NR′C(═O)R″, NR′C(═S)R″, NR′C(═O)OR″, NR′C(═S)OR″, NR′C(═O)SR″, NR′C(═S)SR″, OC(═O)NR′R″, SC(═O)NR′R″, OC(═S)NR′R″, SC(═S)NR′R″, NR′C(═O)NR″R″, and NR′C(═S)NR″R″ with each R′ and each R″ independently being H, aryl or alkyl, and R′″ independently being aryl or alkyl. 
       
     
     
         14 . The dienophile according to  claim 10 , wherein the diene satisfies any one of the formulae: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         15 . The dienophile according to  claim 10 , wherein the diene satisfies any one of the formulae: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         16 . The dienophile according to  claim 10 , wherein the diene satisfies any one of the formulae: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         17 . The dienophile according to  claim 10 , wherein the diene satisfies the formula: 
       
         
           
           
               
               
           
         
       
     
     
         18 . The dienophile according to  claim 10 , wherein the diene satisfies the formula: 
       
         
           
           
               
               
           
         
       
     
     
         19 . The dienophile according to  claim 10 , wherein the diene satisfies the formula: 
       
         
           
           
               
               
           
         
       
     
     
         20 . The dienophile according to  claim 10 , wherein the diene satisfies the formula: